The ATP-binding site of DNA gyrase (on the B subunit) has been located by use of an affinity label. The reagent adenylyl-pyridoxal phosphate labels two lysines at positions 103 and 110 in a region with weak homology to some other ATPases. Another ATP analog, (beta, gamma-imido) ATP, which inhibits DNA gyrase by binding reversibly, has been shown to bind very slowly to the enzyme, in contrast to ATP whose binding is rapid.